Kidney stones are a common and painful human health care problem worldwide. Of all stones developed approximately 75% are composed of calcium oxalate. Urinary oxalate is regarded as a driving force for calcium oxalate stone formation, and absorption of dietary oxalate contributes to 10-70% of urinary oxalate. In fact, roughly 2.4 million Americans (0.75% of the US population) have either enteric or absorptive hyperoxaluria and suffer from recurrent calcium oxalate stone formation. Treatment strategies that significantly reduce the absorption of dietary oxalate and subsequently decrease risk of stone formation are limited if not non- existent. The approach underlying the SBIR Phase I portion of this project was to find an oxalate-degrading enzyme from natural sources (edible fungi) with the ideal properties to be a potential therapeutic, medical food or dietary supplement: stable and active at pH 2.0-5.0, intracellular and/or associated with cell walls, and capable to degrade most available ingested oxalate in the human stomach within the gastric emptying time. Captozyme was successful at finding two enzymes with more than ideal properties. In fact, these enzymes were naturally protected from pepsin degradation, stable at acidic conditions below the pH 2.0 threshold, stable to temperatures approaching 77oC, and the oxalate-degrading enzyme content was successfully increased to 150 units of activity per gram of dry material for one fungal species. Recently, OxDC content from shake flask and fermenter growths was further increased to approximately 600 and 250 units per gram of dry material from one fungal species, respectively, by using unconventional OxDC inducing strategies. The Phase II specific aims are 1) to increase OxDC activity in the dry fungal material obtained from the fermenter process to further reduce dose size, improve patient compliance, and decrease manufacturing costs; 2.) Finish all necessary preparations to establish an early cGMP production process and manufacture five batches at pilot scale; and 3.) Conduct pre-clinical studies to evaluate product safety and demonstrate proof of principle in a dog model. At the conclusion of a successful SBIR Phase II grant Captozyme expects to have all necessary information for a GRAS evaluation and to conduct a clinical trial. The product established by way of this project will ideally be taken with meals and oxalate containing snacks and will likely have a wide application within the calcium oxalate stone- forming population. PUBLIC HEALTH RELEVANCE: Kidney stones are a common and painful human health care problem. In Western countries including the United States, approximately 75% of all stones are composed of calcium oxalate, and roughly a third of these cases originate from recurrent stone formers who experience some degree of elevated urinary oxalate levels (hyperoxaluria). Roughly 2.4 million Americans have either enteric or absorptive hyperoxaluria and are capable of responding to dietary restrictions. Currently there is no treatment strategy that is effective a degrading dietary oxalate and subsequently reducing the recurrence of these kidney stones. Therefore, the overall goal of this project is to develop an oxalate-degrading enzyme that can effectively act as a dietary intercept and hence reduce the occurrence of calcium oxalate stone development in these patient populations.